The long term goal of this project continues to be to unravel the molecular mechanism(s) whereby the insulin receptor tyrosine kinase causes activation of protein serine/threonine kinases. During the previous grant period we have shown that the insulin receptor tyrosine kinase also displays autophosphorylation on ser/thr residues to significant stoichiometry, and we have identified and purified a novel insulin-activated protein serine kinase from rat liver (p90ISK). We also found that overexpression of an activated p21Ras mutant in 3T3-L1 adipocytes mimics insulin's ability to stimulate glucose uptake and translocate glucose transporter. Similarly, recent results from several groups and our shows the proto-oncogene p21Ras is activated (elevated GTP-bound state) by insulin and, when activated, mimics insulin to stimulated protein ser/thr kinases. Thus, a central hypothesis of our work is that p21 couples insulin receptor signal transmission to protein ser/thr kinases. The focus of this proposal is to determine the molecular linkage between the insulin receptor tyrosine kinase and p21Ras regulatory protein involved in p21Ras activation. We shall test the hypothesis that P21Rs activation by insulin is mediated by son-of-sevenless (Sos) proteins, the major known GTP/GDP exchange factors in insulin-sensitive cells, using a newly developed permeabilized 3T3-L1 cell system to directly monitor GTP loading of p21Ras and its hydrolysis. The hypothesis that GTP/GDP exchange activity of Sos proteins in 3T3-L1 adipocytes is modulated by phosphorylation will be evaluated. Insulin action on phosphorylation of drosophila (dSosHA) or mouse (mSos-1HA) Sos proteins tagged with the hemagglutinin (HA) epitope, transiently expressed in COS cells, will also be determined. The functional significance of such phosphorylation will be tested using mutant Sos constructs lacking insulin-regulated phosphorylation sites. We shall also test the alternative hypothesis that insulin causes recruitment of cytosolic Sos proteins to the cell surface membrane where p21Ras resides. Whether insulin stimulates association of Sos proteins with plasma membrane p21Ras will be addressed using chemical crosslinkers. Digital imaging microscopy will assess cellular localization of Sos proteins in intact cells. Finally, based on exciting new data indicating insulin causes an association between Sos GTP/GDP exchange proteins and the p85 regulatory subunit of PI3 kinase, we shall test whether an insulin receptor signaling complex is formed involving these components and IRS-1. The postulated that PPPXPP motifs in the C-terminal domain of Sos proteins govern their binding to SH3 domains encoded in p85 isoforms will be evaluated by mutagenesis and expression of deletion Sos protein constructs. Taken together, theses studies will define underlying mechanisms whereby the Sos GTP/GDP exchange proteins for p21Ras are involved in insulin receptor signal transmission.